Peptides and DNA encoding the peptides useful for immunizations against Coccidioides spp. infections

ABSTRACT

The present invention provides compositions of peptides and polynucleotides encoding the peptides, which peptides are useful for generating an immunological response in an individual and in therapeutic and diagnostic applications of infections due to pathogenic  Coccidioides  spp. fungi, such as  C. immitis  or  C. posadasii.

RELATED APPLICATIONS

This application claims benefit of priority under 35 U.S.C. 119(B) ofProvisional application 60/373,635, filed Apr. 19, 2002, the contents ofwhich are incorporated herein by reference.

GOVERNMENT LICENSE RIGHTS

The United States Government has a paid up license in this invention andthe right in limited circumstances to require the patent owner tolicense others on reasonable terms as provided for by the terms of PHSResearch Grant No. 5 P01 A/37232-06 awarded by the National Institutesof Health.

FIELD OF THE INVENTION

The present invention relates generally to the fields of pathogenicfungi and immunology. More particularly, the present invention providescompositions of Coccidioides spp. peptides and polynucleotides encodingthe peptides, which peptides are useful for generating or detecting animmunological response in an individual and in vaccines and therapeuticapplications of infections due to pathogenic Coccidioides spp. fungi,such as C. posadasii or C. immitis.

BACKGROUND OF THE INVENTION

Coccidioidomycosis, otherwise known as the San Joaquin Valley Fever, isa fungal respiratory disease of humans and wild and domestic animalswhich is endemic to southwestern United States, northern Mexico, andnumerous semiarid areas of Central and South America (Pappagianis, D.Epidemiology of Coccidioidomycosis. Current Topics in Medical Mycology.1988. 2:199–23). Infection occurs by inhalation of airborne spores(arthroconidia) produced by the saprobic phase of Coccidioides spp.,which grows in alkaline desert soil. C. immitis was the first describedspecies, and is now becoming known as the Californian species. The C.posadasii species was recently defined, and was previously recognized asthe non-Californian population of C. immitis (Fisher, M. C., Koenig, G.L., White, T. J., Taylor, J. W. Molecular and phenotypic description ofCoccidioides posadasii sp. nov., previously recognized as thenon-California population of Coccidioides immitis. Mycologia 2002.94(1):73–84, 2002). The differences in the two species are slight.Morphologically they are indistinguishable and no differences in theirability to cause disease are known.

It is estimated that 100,000 new cases of this disease occur annuallywithin the rapidly growing population of people who live in regions ofthe United States between southwest Texas and southern California, wherethe disease is endemic (Galgiani, J. N. Coccidioidomycosis: A regionaldisease of national importance; rethinking our approaches to itscontrol. Annals of Internal Medicine. 1999. 130:293–300). Although themajority of immunocompetent individuals are able to resolve theirCoccidioides spp. infection spontaneously, the level of morbidityassociated even with the primary form of this respiratory mycosiswarrants consideration of a vaccine against the disease.Immunocompromised patients, including those infected with humanimmunodeficiency virus, are at high risk to contract disseminatedcoccidioidomycosis (Ampel, N. M., C. L. Dols, and J. N. Galgiani.Results of a prospective study in a coccidioidal endemic area. AmericanJournal of Medicine. 1993. 94:235–240). It is also apparent from resultsof several clinical studies that African-Americans and Asians aregenetically predisposed to development of the potentially fatal,disseminated form of the respiratory disease (Galgiani, J. N. 1993.Coccidioidomycosis. Western Journal of Medicine 159:153–171).

The rationale for commitment of research efforts to develop aCoccidioides spp. vaccine is based on clinical evidence that individualswho recover from the respiratory coccidioidomycosis disease retaineffective long-term cellular immunity against future infections by thepathogen (Smith, C. E. 1940. American Journal of Public Health30:600–611). In addition, early preclinical studies demonstrated that aformalin-killed whole-cell (spherule) vaccine prevented deaths in miceafter infection with even very large numbers of coccidioidal spores(Levine et al.1961. Journal of Immunology 87:218–227). However, when asimilar vaccine preparation was evaluated in a human trial, there wassubstantial local inflammation, pain, and induration at the injectionsite, rendering the vaccine unacceptable (Pappagianis et al. Evaluationof the protective efficacy of the killed Coccidioides immitis spherulevaccine in humans. American Review of Respiratory Diseases. 1993.148:656–660). Further, there was no difference in the number of cases ofcoccidioidomycosis or the severity of the disease in the formalin-killedspherule vaccinated group compared to the placebo group. Therefore, theoriginal human vaccine trial was not successful.

Subsequent attempts to develop a coccidioidal vaccine focused on crudeor partially purified subcellular preparations from the fungus, and hadlimited success in experimental models (Zimmermann, C. R., S. M.Johnson, G. W. Martens, A. G. White, B. L. Zimmer, and D. Pappagianis.Protection against lethal murine coccidioidomycosis by a soluble vaccinefrom spherules. Infection and Immunity. 1988. 66:2342–2345; Lecara, G.,Cox, R. A., and Simpson, R. B. Coccidioides immitis vaccine: potentialof an alkali-soluble, water-soluble cell wall antigen. Infection andImmunity. 1983. 39: 473–475; Cole, G. T., T. N. Kirkland, and S. H. Sun.An immunoreactive, water-soluble conidial wall fraction of Coccidioidesimmitis 1987. Infection and Immunity 55:657–667; Cole G. T., Kirkland T.N., Franco M., Zhu S., Yuan L., Sun S. H., Hearn V. M. Immunoreactivityof a surface wall fraction produced by spherules of Coccidioidesimmitis. Infection and Immunity October 1988; 56:2695–701).

There is a long felt need for a more effective and usable treatment orvaccination regimen to prevent, treat, or ameliorate infection ofCoccidioides spp. and disease states associated with the infection.

SUMMARY OF THE INVENTION

Accordingly, it is an object herein to provide the methods foridentifying and isolating polypeptides and nucleic acids encodingpolypeptides of Coccidioides spp. that have an immunostimulatoryactivity. Such immunostimulatory nucleotides and polypeptides will beuseful in the prevention, treatment, and diagnosis of infections due toCoccidioides spp.

In order to meet these needs, the present invention providescompositions and methods for the production of antigens comprisingpolypeptide fragments of the Ag2/PRA protein of C. posadasii, includingbut not limited to the polypeptide sequences of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 and or SEQ ID NO:11.

The present invention also provides polynucleotides encoding thepolypeptides, produced by recombinant technology from the Ag2/PRA geneand gene fragments derived from Coccidioides posadasii, including butnot limited to the polynucleotide sequences of SEQ ID NO:3, SEQ ID NO:6,SEQ ID NO:8 and or SEQ ID NO:10.

In one embodiment, the polypeptide of the present invention encompassesthe polypeptide sequence of amino acids 37 to 142 of SEQ ID NO:5 and orthe polypeptide sequence of amino acids 21 to 126 of SEQ ID NO:9, and isreferred to as Ag2/PRA1-106. In another embodiment, the presentinvention includes a polypeptide that lacks the corresponding N-terminalamino acids 1 to 26 of Ag2/PRA1-106 (exclusive of the amino acids of thefusion partner protein encoded by the vector), resulting in thepolypeptide sequence of amino acids 35 to 114 of SEQ ID NO:7, and isreferred to as Ag2/PRA27-106.

The present invention also provides the use of the Ag2/PRA1-106 andAg2/PRA27-106 polypeptides and polynucleotides encoding the polypeptidesto elicit an immune response sufficient to provide an effectiveimmunization against Coccidioides spp. infection. In one embodiment, thepolypeptides provide protection against Coccidioides posadasii and orCoccidioides immitis infections in a mammal, such as a human. In anotherembodiment, the polypeptides provide protection against Coccidioidesspp. infection in domestic animals, including but not limited to dogs,cats, horses, and cattle. In a further embodiment, the inventionprovides polynucleotides encoding Ag2/PRA1-106 and or Ag2/PRA27-106polypeptides in a vector suitable for transforming mammalian cells as amethod for immunizing mammals against Coccidioides spp. infection.

The present invention further provides compositions and the methods ofuse of the above-mentioned Ag2/PRA1-106 and Ag2/PRA27-106 polypeptidesin combination with one or more other Coccidioides spp. antigens toelicit an immune response sufficient to provide an effectiveimmunization against Coccidioides spp. infection. In one embodiment thepolypeptides are provided as a composition containing a mixture of saidantigens, for example, Ag2/PRA1-106 and Coccidioides-immitis specificantigen (referred to hereafter as Csa), (Pan, S. and Cole, G. T. 1995.Molecular and biochemical characterization of Coccidioidesimmitis-specific (CS) antigen. Infection and Immunity, 63:3994–4002). Inanother embodiment the composition is provided as a single fusionpolypeptide comprised of Coccidioides spp. antigens, for example theAg2/PRA1-106+Csa chimeric fusion polypeptide of SEQ ID NO:11.

The invention also provides expression vectors that include regulatorysequences such as promoters or other transcriptional regulatory elementsoperably linked to the nucleotide sequences that control expression ofthe nucleotide sequences or degenerate variants of the sequences in hostcells for the production of the polypeptides of SEQ ID NO:5, or SEQ IDNO:7, or SEQ ID NO:9, or SEQ ID NO:11.

The invention further provides host cells derived from yeast, bacterial,plant, animal or human sources containing the expression vectorscomprising the sequences of SEQ ID NO:3 or SEQ ID NO:6 or SEQ ID NO:8 orSEQ ID NO:10.

The present invention also provides shorter polypeptide fragmentsincluded within amino acids 37 to 142 of SEQ ID NO:5 and or amino acids55 to 136 of SEQ ID NO:5 and or amino acids 21 to 126 of SEQ ID NO:9 andor amino acids 35 to 114 of SEQ ID NO:7 and or amino acids 11 to 264 ofSEQ ID NO:11, respectively. In preferred embodiments, these shorterpolypeptides may include polypeptides of not less than 25 amino acids inlength, inclusive of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, and 78–100 amino acids in length. Such polypeptidefragments, which are substantially the same amino acid length as thesequences containing the antigenic epitopes, provide similar ability toelicit an immune response, including such immune responses that providesprotection against Coccidioides spp. infection.

In another embodiment, the present invention includes polypeptides whichare substantially identical to the polypeptide sequences of SEQ ID NO:5and or SEQ ID NO:8 and or SEQ ID NO:7 and or SEQ ID NO:11 and or containat least one conservative amino acid substitution. Such polypeptides,which are substantially the same amino acid length, provide similarability to elicit an immune response, including such immune responsesthat provides protection against Coccidioides spp. infection. Suchpolypeptides, having substantial identity to the polypeptides ofAg2/PRA1-106 and or Ag2/PRA27-106, include those polypeptides at leastabout 99% identical or equivalent, at least about 95% identical orequivalent, at least about 90% identical or equivalent, at least about85% identical or equivalent, at least about 80% identical or equivalent,at least about 75% identical or equivalent, and at least about 70%identical to said polypeptides and which have the aforementionedactivities, are encompassed in the invention.

The present invention further provides methods and compositions ofisolated polypeptides identical or substantially identical to thepolypeptide sequences of SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:7 and orSEQ ID NO:11 useful in pharmaceutical compositions.

The present invention also provides vaccine formulations and methods ofpreparing the formulations containing the Ag2/PRA1-106 and orAg2/PRA27-106 polypeptides and or polynucleotides encoding thepolypeptides. The present invention further provides vaccineformulations containing adjuvants and pharmaceutical excipients andcarriers.

The present invention provides the Ag2/PRA1-106 and or Ag2/PRA27-106 andor other Coccidioides spp. polypeptides as vaccine formulations andmethods for eliciting an effective immune response in a mammal,including humans and domestic animals, for the prevention ofCoccidioides spp. infections.

The present invention further provides kits containing the Ag2/PRA1-106and or Ag2/PRA27-106 polypeptides and or other Coccidioides spp.antigens and or polynucleotides encoding the polypeptides, to facilitatethe use of the polypeptides and or polynucleotides.

The present invention also provides a contruct comprising a promotersequence for the Ag2/PRA1-106 and or Ag2/PRA27-106 genes encoding thepolypeptides and or other Coccidioides spp. antigens, which can directgene expression in a host cell.

The present invention also provides the use of the Ag2/PRA1-106 and orAg2/PRA27-106 polypeptides and or polynucleotides encoding theAg2/PRA1-106 and or Ag2/PRA27-106 polypeptides in diagnostic kits forthe detection of infections due to Coccidioides spp. in mammals, such ashumans and domestic animals.

The present invention also provides an antibody specific for an antigenof the Ag2/PRA1-106 and or Ag2/PRA27-106 polypeptides and methods forthe creation of such antibodies. Such antibodies may be used indiagnostic kits for the detection of infections due to Coccidioides spp.The present invention provides kits containing antibodies in suitablecompositions for the detection of infections due to Coccidioides spp. inmammals, such as humans and domestic animals.

The above and other aspects of the invention will become readilyapparent to those of skill in the art from the following detaileddescription and figures, wherein only the preferred embodiments of theinvention are shown and described, simply by way of illustration of thebest mode of carrying out the invention. As is readily recognized, theinvention is capable of modifications within the skill of the relevantart without departing from the spirit and scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: Nucleotide (SEQ ID NO:1) and derived amino acid sequences (SEQID NO:2) of the cDNA C. posadasii Ag2/PRA gene aligned by the TRANSLATEprogram of the GCG Package. The translated nucleotide sequence resultsin a protein of 194 amino acid residues with a putative N-terminalsignal sequence as the first 18 amino acids.

FIG. 2: The aligned nucleotide (SEQ ID NO:3) and deduced amino acidsequences (SEQ ID NO:4) of the recombinant Ag2/PRA1-106 gene expressedin an E. coli host. The translated nucleotide sequence results in aprotein of 271 amino acid residues, of which 142 are retained afterthrombin cleavage (SEQ ID NO:5).

FIG. 3: The aligned nucleotide (SEQ ID NO:6) and deduced amino acidsequences (SEQ ID NO:7) of the recombinant Ag2/PRA27-106 gene. Thetranslated nucleotide sequence results in a protein of 114 amino acidresidues.

FIG. 4: The aligned nucleotide (SEQ ID NO:8) and deduced amino acidsequences (SEQ ID NO:9) of the recombinant Ag2/PRA1-106 gene expressedin a Saccharomyces cerevisiae host. The translated nucleotide sequenceresults in a protein of 126 residues.

FIG. 5: Annotated diagram of full-length Ag2/PRA and subunits evaluatedin experimental mouse models of coccidioidomycosis.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO:1 depicts the determined cDNA nucleotide sequence encodingfull-length Ag2/PRA;

SEQ ID NO:2 depicts the deduced amino acid sequence of the full-lengthAg2/PRA polypeptide encoded by the nucleotide sequence of SEQ ID NO:1;

SEQ ID NO:3 depicts the determined nucleotide sequence of the pET32arecombinant construct encoding the Ag2/PRA1-106 fusion polypeptide;

SEQ ID NO:4 depicts the deduced amino acid sequence of the recombinantAg2/PRA1-106 fusion polypeptide, including 165 N-terminal amino acidsderived from the pET32a vector, encoded by the nucleotide sequence ofSEQ ID NO:3 and produced in E. coli;

SEQ ID NO:5 depicts the deduced amino acid sequence of the recombinantAg2/PRA1-106 fusion polypeptide remaining after thrombin cleavage of thepolypeptide of SEQ ID NO:4, including 36 N-terminal amino acids (SEQ IDNO:20) derived from the pET32a vector;

SEQ ID NO:6 depicts the determined nucleotide sequence of the pET28arecombinant construct encoding the Ag2/PRA27-106 fusion polypeptide;

SEQ ID NO:7 depicts the deduced amino acid sequence of the recombinantAg2/PRA27-106 fusion polypeptide, including 34 N-terminal amino acids(SEQ ID NO: 21) derived from the pET28a vector, encoded by thenucleotide sequence of SEQ ID NO:5 and produced in E. coli;

SEQ ID NO:8 depicts the determined nucleotide sequence of the YEpFLAG-1recombinant construct encoding the Ag2/PRA1-106 fusion polypeptide;

SEQ ID NO:9 depicts the deduced amino acid sequence of the recombinantAg2/PRA1-106 fusion polypeptide, including 20 N-terminal amino acids(SEQ ID NO:22) derived from the YEpFLAG-1 vector, encoded by thenucleotide sequence of SEQ ID NO:8 and produced in Saccharomycescerevisiae;

SEQ ID NO:10 depicts the determined nucleotide sequence of the YEpFLAG-1recombinant construct encoding the Ag2/PRA1-106+Csa chimeric fusionpolypeptide;

SEQ ID NO:11 depicts the deduced amino acid sequence of the recombinantAg2/PRA1-106+Csa chimeric fusion polypeptide, including 10 N-terminalamino acids and two amino acids separating Ag2/PRA1-106 and Csa derivedfrom the vector, encoded by the nucleotide sequence of SEQ ID NO:10 andproduced in Saccharomyces cerevisiae;

SEQ ID NO:12 depicts the nucleotide sequence of the sense P1 primer usedfor cloning the Ag2/PRA sequence into the pET32a and pVR1020 vectors andsubcloning the Ag2/PRA1-106 sequence into the pET32a, pVR1020 andYEpFLAG-1 vectors at the BamHl restriction site;

SEQ ID NO:13 depicts the nucleotide sequence of the antisense P2 primerused for cloning the Ag2/PRA sequence into the pET32a vector at theEcoRI restriction site;

SEQ ID NO:14 depicts the nucleotide sequence of the antisense P10 primerused for cloning the Ag2/PRA sequence into the pVR1020 vector at theBglII restriction site;

SEQ ID NO:15 depicts the nucleotide sequence of the antisense P4 primerused to subclone the Ag2/PRA1-106 sequence into the pET32a vector andthe Ag2/PRA27-106 sequence into the pET28a vector at the EcoRIrestriction sites;

SEQ ID NO:16 depicts the nucleotide sequence of the sense P3 primer usedto subclone the Ag2/PRA27-106 sequence into the pET28a vector at theBamHI restriction site;

SEQ ID NO:17 depicts the nucleotide sequence of the antisense P8 primerused to subclone the Ag2/PRA1-106 sequence into the pVR1020 vector atthe BglII restriction site;

SEQ ID NO:18 depicts the nucleotide sequence of the sense P5 primer usedto subclone the Ag2/PRA27-106 sequence into the pVR1020 vector at theBamHI restriction site;

SEQ ID NO:19 depicts the nucleotide sequence of the antisense P11 primerused to subclone the Ag2/PRA1-106 sequence into the YEpFLAG-1 vector atthe SalI restriction site;

SEQ ID NO: 20 depicts the deduced amino acid sequence of the fusionpartner peptide derived from the pET-32a vector at the N-terminal of therecombinant Ag2/PRA1-106 produced in E. coli;

SEQ ID NO: 21 depicts the deduced amino acid sequence of the fusionpartner peptide derived from the pET-28a vector at the N-terminal of therecombinant Ag2/PRA27-106 produced in E. coli;

SEQ ID NO:22 depicts the deduced amino acid sequence of the fusionpartner peptide derived from the YEpFLAG-1 vector at the N-terminal ofthe recombinant Ag2/PRA1-106 produced in Saccharomyces cerevisiae;

SEQ ID NO:23 depicts the determined cDNA sequence encoding the Csapolypeptide;

SEQ ID NO:24 depicts the nucleotide sequence of the synthetic CpGadjuvant used in animal experiments.

SEQ ID NO:25 depicts the nucleotide sequence of the sense P14 primerused for cloning the Ag2/PRA1-106 sequence component of theAg2/PRA1-106+Csa chimeric construct into the YEpFLAG-1 vector at theEcoRI restriction site;

SEQ ID NO:26 depicts the nucleotide sequence of the sense P15 primerused for cloning the Ag2/PRA1-106 sequence component of theAg2/PRA1-106+Csa chimeric construct into the YEpFLAG-1 vector at theEcoRI restriction site;

SEQ ID NO:27 depicts the nucleotide sequence of the sense P16 primerused for cloning the CSA sequence component of the Ag2/PRA1-106+Csachimeric construct into the YEpFLAG-1 vector at the BamHI restrictionsite; and

SEQ ID NO:28 depicts the nucleotide sequence of the sense P17 primerused for cloning the CSA sequence component of the Ag2/PRA1-106+Csachimeric construct into the YEpFLAG-1 vector at the SalI restrictionsite.

SEQ ID NO:29 depicts the nucleotide sequence of the sense P5 primer usedto subclone the Ag2/PRA90-151 and Ag2/PRA90-194 sequences into thepET28a vector at the BamHI restriction site;

SEQ ID NO:30 depicts the nucleotide sequence of the antisense P6 primerused to subclone the Ag2/PRA90-151 sequence into the pET28a vector atthe EcoRI restriction site and the sense P6 primer used to subclone theAg2/PRA90-194 sequence into the pVR1020 vector at the EcoRI restrictionsite;

SEQ ID NO:31 depicts the nucleotide sequence of the antisense P7 primerused to subclone the Ag2/PRA90-194 sequence into the pET28a vector atthe EcoRI restriction site;

SEQ ID NO:32 depicts the nucleotide sequence of the antisense P9 primerused to subclone the Ag2/PRA90-151 sequence into the pET28a vector atthe BglII restriction site;

SEQ ID NO:33 depicts the nucleotide sequence of the antisense P10 primerused to subclone the Ag2/PRA1-194 and Ag2/PRA90-194 sequences into thepVR1020 vector at the BglII restriction site;

SEQ ID NO:34 depicts the nucleotide sequence of the sense P12 primerused to subclone the Ag2/PRA19-100 sequence into the pET32a vector atthe BamHI restriction site; and

SEQ ID NO:35 depicts the nucleotide sequence of the antisense P13 primerused to subclone the Ag2/PRA19-100 sequence into the pET32a vector atthe EcoRI restriction site.

DETAILED DESCRIPTION OF THE INVENTION

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described herein. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In addition, the materials,methods, and examples are illustrative only and are not intended to belimiting.

Reference is made to standard textbooks of molecular biology thatcontain definitions and methods and means for carrying out basictechniques, encompassed by the present invention. See, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition,Cold Spring Harbor Laboratory Press, New York (2001), Current Protocolsin Molecular Biology, Ausubel et al (eds.), John Wiley & Sons, New York(2001) and the various references cited therein.

I. The Polypeptide Sequences of the Invention.

The invention focuses on the use of Ag2/PRA polypeptide fragments andthe nucleotide sequences that encode them as immunogenic antigens for apreventative or therapeutic vaccine for coccidioidomycosis, or fordetection of immune responses in individuals infected by Coccidioidesspp.

Native Ag2/PRA is a 194 amino acid, proline-rich protein which is acomponent of a glycopeptide identified originally as “antigen 2” by atwo-dimensional electrophoresis classification scheme (Huppert et al.1978. Infection and Immunity 20:541–551). It contains an N-terminalsignal sequence of 18 amino acids. The amino acid sequence of nativeAg2/PRA is shown in SEQ ID NO:2. The genomic sequence was cloned andsubsequently modified to produce select polypeptide fragments andshorter DNA vaccine constructs. The reasons for reducing the full-lengthantigen to fragments are multiple; 1) a positive correlation betweendevelopment of DTH (delayed-type hypersensitivity) (a Th1 immuneresponse) to coccidioidal antigens and the ability to resistdisseminated coccidioidomycosis has been shown. This response isgenerally regarded as a MHC II type response (Louie et al. 1999.Influence of host genetics on the severity of coccidioidomycosis.Emerging Infectious Diseases 5:672–680) and that peptides that bind toMHC II receptors are generally 13–25 residues in length; 2)determination of epitopes within a larger polypeptide or multiplepeptides that enhance or suppress an immune response allow for thecreation of fusion polypeptides containing select epitopes that can beused as vaccines with increased potency (Sette A, et al. 2002.Optimizing vaccine design for cellular processing, MHC binding and TCRrecognition. Tissue Antigens 59:443–451) and 3) reducing the size of thepolypeptide can lead to desirable advantages in the production,purification and safety of a vaccine. Consequently, the full-lengthAg2/PRA polypeptide sequence was evaluated and recombinant methods wereused to create nucleotide sequences and polypeptides that encoded orcontained regions with predicted antigenic motifs and were evaluated fortheir ability to elicit a protective immune response. The rAg2/PRA1-106of the present invention is a polypeptide of 106 amino acids in length,retaining the N-terminal signal sequence of 18 amino acids found innative Ag2/PRA. In the case of the rAg2/PRA1-106 of the presentinvention produced in E. coli, it was initially expressed as a fusionpolypeptide in the E. coli host transformed with the Ag2/PRA1-106-pET32aplasmid construct, resulting in a recombinant fusion polypeptide with271 amino acids (SEQ ID NO:4). The aforementioned fusion polypeptide wassubsequently treated with thrombin to release a polypeptide of 142 aminoacids (SEQ ID NO:5), of which amino acids 37 to 142 of the sequencerepresent rAg2/PRA1-106 of the present invention. In the case of therAg2/PRA1-106 of the present invention produced in Saccharomycescerevisiae, it was expressed as a fusion polypeptide in the yeast hosttransformed with the Ag2/PRA1-106-YEpFLAG-1 plasmid construct, resultingin a recombinant fusion polypeptide with 126 amino acids (SEQ ID NO:9),of which amino acids 21 to 126 represent rAg2/PRA1-106 of the presentinvention.

The rAg2/PRA27-106 of the present invention is expressed as a fusionpolypeptide of 114 amino acids in length, a construct designed toeliminate the N-terminal signal sequence of the native polypeptide. Theamino acid sequence of the rAg2/PRA27-106 fusion polypeptide produced inthe E. coli host transformed with the Ag2/PRA27-106-pET28a construct isshown in SEQ ID NO:7, of which amino acids 35 to 114 representrAg2/PRA27-106 of the present invention.

Additional polypeptides are encompassed in the invention, consistingessentially of the sequences of the Ag2/PRA1-106 and or Ag2/PRA27-106polypeptides, including polypeptides at least about 99% identical orequivalent, at least about 95% identical or equivalent, at least about90% identical or equivalent, at least about 85% identical or equivalent,at least about 80% identical or equivalent, at least about 75% identicalor equivalent, and at least about 70% identical to the sequences of therespective polypeptides.

As used herein, the terms “protein” or “polypeptide” are used in thebroadest sense to mean a sequence of amino acids that can be encoded bya cellular gene or by a recombinant nucleic acid sequence or can bechemically synthesized. In some cases, the term “polypeptide” is used inreferring to a portion of an amino acid sequence (peptides) of afull-length protein. An active fragment of a Ag2/PRA is an example ofsuch a polypeptide. A protein can be a complete, full-length geneproduct, which can be a core protein having no amino acid modifications,or can be a post-translationally modified form of a protein such as aphosphoprotein, glycoprotein, proteoglycan, lipoprotein ornucleoprotein.

“Consisting essentially of”, in relation to amino acid sequence of apolypeptide, protein or peptide, is a term used hereinafter for thepurposes of the specification and claims to refer to a conservativesubstitution or modification of one or more amino acids in that sequencesuch that the tertiary configuration of the polypeptide, protein orpeptide is substantially unchanged.

“Conservative substitutions” is defined by substitutions of amino acidshaving substantially the same charge, size, hydrophilicity, and oraromaticity as the amino acid replaced. Such substitutions, known tothose of ordinary skill in the art, include glycine-alanine-valine;isoleucine-leucine; tryptophan-tyrosine; aspartic acid-glutamic acid;arginine-lysine; asparagine-glutamine; and serine-threonine.

“Modification”, in relation to amino acid sequence of a polypeptide,protein or peptide, is defined functionally as a deletion of one or moreamino acids which does not impart a change in the conformation, andhence the biological activity, of the polypeptide, protein or peptidesequence.

The common amino acids are generally known in the art. Additional aminoacids that may be included and or substituted in the peptide of thepresent invention include: L-norleucine; aminobutyric acid;L-homophenylalanine; L-norvaline; D-alanine; D-cysteine; D-asparticacid; D-glutamic acid; D-phenylalanine; D-histidine; D-isoleucine;D-lysine; D-leucine; D-methionine; D-asparagine; D-proline; D-glutamine;D-arginine; D-serine; D-threonine; D-valine; D-tryptophan; D-tyrosine;D-omithine; aminoisobutyric acid; L-ethylglycine; L-t-butylglycine;penicillamine; I-naphthylalanine; cyclohexylalanine; cyclopentylalanine;aminocyclopropane carboxylate; aminonorbornylcarboxylate;L-α-methylalanine; L-α-methylcysteine; L-α-methylaspartic acid;L-α-methylglutamic acid; L-α-methylphenylalanine; L α-methylhistidine;L-α-methylisoleucine; L-α-methyllysine; L-α-methylleucine;L-α-methylmethionine; L-α-methylasparagine; L-α-methylproline;L-α-methylglutamine; L-α-methylarginine; L-α-methylserine;L-α-methylthreonine; L-α-methylvaline; L-α-methyltryptophan;L-α-methyltyrosine; L-α-methylomithine; L-α-methylnorleucine;amino-α-methylbutyric acid;. L-α-methylnorvaiine;L-α-methylhomophenylalanine; L-α-methylethylglycine;methyl-γ-aminobutyric acid; methylaminoisobutyric acid;L-α-methyl-t-butylglycine; methylpenicillamine;methyl-α-naphthylalanine; methylcyclohexylalanine;methylcyclopentylalanine; D-α-methylalanine; D-α-methylornithine;D-α.-methylcysteine; D-α-methylaspartic acid; D-α-methylglutamic acid;D-α-methylphenylalanine; D-α-methylhistidine; D-α-methylisoleucine;D-α-methyllysine; D-α-methylleucine; D-α-methylmethionine;D-α-methylasparagine; D-α-methylproline; D-α-methylglutamine;D-α-methylarginine; D-α-methylserine; D-α-methylthreonine;D-α-methylvaline; D-α-methyltryptophan; D-α-methyltyrosine;L-N-methylalanine; L-N-methylcysteine; L-N-methylaspartic acid;L-N-methylglutamic acid; L-N-methylphenylalanine; L-N-methylhistidine;L-N-methylisoleucine; L-N-methyllysine; L-N-methylleucine;L-N-methylmethionine; L-N-methylasparagine; N-methylcyclohexylalanine;L-N-methylglutamine; L-N-methylarginine; L-N-methylserine;L-N-methylthreonine; L-N-methylvaline; L-N-methyltryptophan;L-N-methyltyrosine; L-N-methylomithine; L-N-methylnorleucine;N-amino-α-methylbutyric acid; L-N-methylnorvaline;L-N-methylhomophenylalanine; L-N-methylethylglycine;N-methyl-γaminobutyric acid; N-methylcyclopentylalanine;L-N-methyl-t-butylglycine; N-methylpenicillamine;N-methyl-α-naphthylalanine; N-methylaminoisobutyric acid;N-(2-aminoethyl)glycine; D-N-methylalanine; D-N-methylornithine;D-N-methylcysteine; D-N-methylaspartic acid; D-N-methylglutamic acid;D-N-methylphenylalanine; D-N-methylhistidine; D-N-methylisoleucine;D-N-methyllysine; D-N-methylleucine; D-N-methylmethionine;D-N-methylasparagine; D-N-methylproline; D-N-methylglutamine;D-N-methylarginine; D-N-methylserine; D-N-methylthreonine;D-N-methylvaline; D-N-methyltryptophan; D-N-methyltyrosine;N-methylglycine; N-(carboxymethyl)glycine; N-(2-carboxyethyl)glycine;N-benzylglycine; N-(imidazolylethyl)glycine; N-(1-methylpropyl)glycine;N-(4-aminobutyl)glycine; N-(2-methylpropyl)glycine;N-(2-methylthioethyl)glycine; N-(hydroxyethyl)glycine;N-(carbamylmethyl)glycine; N-(2-carbamylethyl)glycine;N-(1-methylethyl)glycine; N-(3-guanidinopropyl)glycine;N-(3-indolylethyl)glycine; N-(p-hydroxyphenethyl)glycine;N-(1-hydroxyethyl)glycine; N-(thiomethyl)glycine;N-(3-aminopropyl)glycine; N-cyclopropylglycine; N-cyclobutyglycine;N-cyclohexylglycine; N-cycloheptylglycine; N-cyclooctylglycine;N-cyclodecylglycine; N-cycloundecylglycine; N-cyclododecylglycine;N-(2,2-diphenylethyl)glycine; N-(3,3-diphenylpropyl)glycine;N-(N-(2,2-diphenylethyl)carbamylmethyl)glycine;N-(N-(3,3-diphenylpropyl)carbamylmethyl)glycine; and1-carboxy-1-(2,2-diphenylethylamino)cyclopropane.

The polypeptides of the present invention can be produced by knownchemical synthesis methods; for example, a liquid phase synthesismethod, a solid phase synthesis method, and others (Izumiya, N., Kato,T., Aoyagi, H., Waki, M., Basis and Experiments of Peptide Synthesis,1985, Maruzen Co., Ltd.).

The polypeptides of the present invention may contain one or moreprotected amino acid residues. The protected amino acid is an amino acidwhose functional group or groups is/are protected with a protectinggroup or groups by a known method or by the use of various protectedamino acids that are commercially available.

Because native Ag2/PRA obtained from Coccidioides spp. is glycosylated,the polypeptides of the present invention may be provided in aglycosylated as well as an unglycosylated form. Preparation ofglycosylated protein or peptide is known in the art and typicallyinvolves expression of the recombinant DNA encoding the protein orpeptide in a eukaryotic cell (e.g., yeast or mammalian). Likewise, it isgenerally known in the art to express the recombinant DNA encoding theprotein or peptide in a prokaryotic (e.g., bacterial) cell to obtain aprotein or peptide, which is not glycosylated. These and other methodsof altering carbohydrate moieties on glycoproteins is found, inter alia,in Essentials of Glycobiology (1999), Edited By Ajit Varki, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., the contents of whichare incorporated herein by reference.

II. The DNA Sequences of the Invention.

Alternatively, the polypeptides of the present invention can be producedby producing a polynucleotide (DNA or RNA) which encodes the amino acidsequence of a polypeptide of the present invention and producing saidpolypeptide by a genetic engineering technique using the polynucleotide.Polynucleotide coding sequences for amino acid residues are known in theart and are disclosed for example in Molecular Cloning: A LaboratoryManual, Third Edition, Sambrook, Fritsch, and Maniatis, Cold SpringHarbor Laboratory Press, 2001.

Analysis of genomic DNA encoding Ag2/PRA reveals an open reading framefor 194 amino acids, interrupted by two introns (GenBank AccessionAF013256). The cDNA sequence encoding full-length Ag2/PRA is shown inSEQ ID NO:1 (GenBank Accession U39835). The nucleotide sequence encodingthe fusion polypeptide rAg2/PRA1-106 of the present invention expressedin the E. coli host is comprised of 816 nucleotides, including a stopcodon (SEQ ID NO:3), in a pET32a plasmid construct(Ag2/PRA1-106-pET32a). Expression of this construct results in arecombinant fusion polypeptide with 271 amino acids (SEQ ID NO:4),including 165 N-terminal fusion partner amino acids derived from thevector.

The nucleotide sequence encoding the fusion polypeptide rAg2/PRA1-106 ofthe present invention expressed in the Saccharomyces cerevisiae host iscomprised of 381 nucleotides, including a stop codon (SEQ ID NO:8), in aYEpFLAG-1 plasmid construct (Ag2/PRA1-106-YEpFLAG-1). Expression of thisconstruct results in a recombinant fusion polypeptide with 126 aminoacids (SEQ ID NO:9), including 20 N-terminal fusion partner amino acidsderived from the YEpFLAG-1 vector. The nucleotide sequence encoding thefusion polypeptide rAg2/PRA27-106 of the present invention in the E.coli host is comprised of 345 nucleotides, including a stop codon (SEQID NO:6), in a pET28a plasmid construct (Ag2/PRA27-106-pET28a).Expression of this construct results in a recombinant polypeptide with114 amino acids (SEQ ID NO:7), which includes 34 N-terminal fusionpartner amino acids derived from the pET28a vector.

Within the context of the present invention “polynucleotide” in generalrelates to polyribonucleotides and polydeoxyribonucleotides, it beingpossible for these to be non-modified RNA or DNA or modified RNA or DNA.Polynucleotides which encode the peptides of the present invention meanthe sequences exemplified in this application as well as those whichhave substantial identity to those sequences and which encode thepeptides. Preferably, such polynucleotides are those which hybridizeunder stringent conditions as defined herein and are at least 70%,preferably at least 80% and more preferably at least 90% to 95%identical to those sequences.

“Consisting essentially of”, in relation to a nucleic acid sequence, isa term used hereinafter for the purposes of the specification and claimsto refer to sequences of the present invention and sequences withsubstitution of nucleotides as related to third base degeneracy. Asappreciated by those skilled in the art, because of third basedegeneracy, almost every amino acid can be represented by more than onetriplet codon in a coding nucleotide sequence. Further, minor base pairchanges may result in variation (conservative substitution) in the aminoacid sequence encoded, which are not expected to substantially alter thebiological activity of the gene product. Thus, a nucleic acid sequencingencoding a protein or peptide as disclosed herein, may be modifiedslightly in sequence (e.g., substitution of a nucleotide in a tripletcodon), and yet still encode its respective gene product of the sameamino acid sequence.

The terms “stringent conditions” or “stringent hybridization conditions”include reference to conditions under which a polynucleotide willhybridize to its target sequence, to a detectably greater degree thanother sequences (e.g., at least 2-fold over background). In particular,a DNA or polynucleotide molecule which hybridizes under stringentconditions is preferably at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%or 99% identical to the DNA that encodes the amino acid sequencesdescribed herein. In a preferred embodiment these polynucleotides thathybridize under stringent conditions also encode a protein or peptidewhich upon administration to a subject provides an immunostimulationsufficient to provide some level of immune protection againstCoccidioides spp. infection as described herein.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short polynucleotides (e.g., 10to 50 nucleotides) and at least about 60° C. for long polynucleotides(e.g., greater than 50 nucleotides)—for example, “stringent conditions”can include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 0.1×SSC at 60 to 65° C.

Homology, sequence similarity or sequence identity of nucleotide oramino acid sequences may be determined conventionally by using knownsoftware or computer programs such as the BestFit or Gap pairwisecomparison programs (GCG Wisconsin Package, Genetics Computer Group, 575Science Drive, Madison, Wis. 53711). BestFit uses the local homologyalgorithm of Smith and Waterman (Advances in Applied Mathematics. 1981.2: 482–489), to find the best segment of identity or similarity betweentwo sequences. Gap performs global alignments: all of one sequence withall of another similar sequence using the method of Needleman andWunsch, (Journal of Molecular Biology. 1970. 48:443–453). When using asequence alignment program such as BestFit to determine the degree ofsequence homology, similarity or identity, the default setting may beused, or an appropriate scoring matrix may be selected to optimizeidentity, similarity or homology scores. Similarly, when using a programsuch as BestFit to determine sequence identity, similarity or homologybetween two different amino acid sequences, the default settings may beused, or an appropriate scoring matrix, such as blosum45 or blosum80,may be selected to optimize identity, similarity or homology scores.

Naturally, the present invention also encompasses DNA segments thatconsist essentially of or are complementary, or essentiallycomplementary, to the sequences set forth in SEQ ID NO:3 or SEQ ID NO: 6or SEQ ID NO:8 or SEQ ID NO:10. Nucleic acid sequences that are“complementary” are those that are capable of base-pairing according tothe standard Watson-Crick complementarity rules. As used herein, theterm “complementary sequences” means nucleic acid sequences that aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segments SEQ ID NO:3 or SEQ ID NO:6 orSEQ ID NO:8 or SEQ ID NO:10 under stringent conditions such as thosedescribed herein.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other nucleicacid and DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid segmentor fragment of almost any length may be employed, with the total lengthpreferably being limited by the ease of preparation and use in theintended recombinant protocol.

For example, nucleic acid segments or fragments may be prepared thatinclude a short contiguous stretch identical to or complementary to theantigen encoding regions of SEQ ID NO:3 or SEQ ID NO:6 or SEQ ID NO:8 orSEQ ID NO:10, such as about a 15, 18 or 21 nucleotide stretch, up toabout 20,000, about 10,000, about 5,000 or about 3,000 base pairs inlength. Nucleic acid and DNA segments with total lengths of about 1,000,about 500, about 200, about 100 and about 50 base pairs in length(including all intermediate lengths) are also contemplated to be useful.

It will be readily understood that “intermediate lengths”, in thesecontexts, means any length between the quoted ranges, such as 21, 22,23, 24, 25, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102,103, etc.; 150, 151, 152, 153, etc.; including all integers through the200–500; 500–1,000; 1,000–2,000; 2,000–3,000; 3,000–5,000; 5,000–10,000ranges, up to and including sequences of about 12,001, 12,002, 13,001,13,002, 15,001, 20,001 and the like.

It will also be understood that this invention is not limited to theparticular nucleic acid and amino acid sequences of SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10 or SEQ ID NO:11, respectively. Recombinant vectors andisolated DNA segments may therefore variously include the coding regionfrom SEQ ID NO:3 or SEQ ID NO: 6 or SEQ ID NO:8 or SEQ ID NO:10 codingregions bearing selected alterations or modifications in the basiccoding region, or they may encode larger polypeptides that neverthelessinclude such coding regions or may encode biologically functionalequivalent proteins or peptides that have variant amino acids sequences.

The nucleic acid and DNA segments of the present invention furtherinclude sequences that encode biologically functional equivalentCoccidioides spp. peptides that arise as a consequence of codonredundancy and functional equivalency that are known to occur naturallywithin nucleic acid sequences and the proteins thus encoded. Equally,functionally equivalent proteins or peptides may be created via theapplication of recombinant DNA technology in which changes in theprotein structure may be engineered, based on considerations of theproperties of the amino acids being exchanged. Changes designed by humanintervention may be introduced through the application of site-directedmutagenesis techniques, e.g., to introduce improvements to theantigenicity of the protein.

III. Expression Vectors, Hosts, and Expression of Polypeptides of theInvention in vitro and in vivo.

The term “expression vector” refers to a polynucleotide that includescoding sequences that encode the polypeptide of the invention andprovides the sequences necessary for its expression in the selected hostcell. Expression vectors will generally include a transcriptionalpromoter and terminator, or will provide for incorporation adjacent toan endogenous promoter. Promoters that are commonly used in recombinantDNA construction include the β-lactamase (penicillinase),β-galactosidase and tryptophan (trp) promoter systems. While these arethe most commonly used, other microbial promoters have been discoveredand utilized, and details concerning their nucleotide sequences havebeen published, enabling those of skill in the art to ligate themfunctionally with plasmid vectors.

The recombinant host cells of the present invention may be maintained invitro, e.g., for recombinant protein, polypeptide or peptide production.Equally, the recombinant host cells could be host cells in vivo, such asresults from immunization of an animal or human with a nucleic acidsegment of the invention. Accordingly, the recombinant host cells may beprokaryotic or eukaryotic host cells, such as E. coli, Saccharomycescerevisiae or other yeast, mammalian or human or plant host cells. Itwill be further appreciated by the skilled practitioner that otherprokaryotic and eukaryotic cells and cell lines may be appropriate for avariety of purposes; e.g., to provide higher expression, desirableglycosylation patterns, or other features. Expression vectors willusually be plasmids, further comprising an origin of replication and oneor more selectable markers. The pET32a-Ag2/PRA1-106 construct of thepresent invention is an example of such expression vectors. AYEpFLAG-1-Ag2/PRA1-106 construct is another example. However, expressionvectors may alternatively be viral recombinants designed to infect thehost, or integrating vectors designed to integrate at a preferred sitewithin the host's genome. Examples of other expression vectors aredisclosed in Molecular Cloning: A Laboratory Manual, Third Edition,Sambrook, Fritsch, and Maniatis, Cold Spring Harbor Laboratory Press,2001.

Such polynucleotides encoding the polypeptides of the invention andexpression vectors carrying the vectors can be used to produce thepolypeptides in vitro or in vivo. The polypeptides so produced can beisolated according to the procedures described herein and commonly knownin the art and then used in a therapeutic or immunization protocol.

One may also prepare fusion proteins and peptides, e.g., where theCoccidioides spp. peptide coding region is included within the sameexpression unit with other proteins or peptides having desiredfunctions, such as for purification or immunodetection purposes (e.g.,proteins that may be isolated by affinity chromatography and enzymelabel coding regions, respectively), or proteins and peptides encodingadditional antigens capable of eliciting a second or enhancedimmunostimulatory response in a subject (e.g., such as the Coccidioidesspp. antigens Csa [SEQ ID NO:23], Gel1, Ure, or non-Coccidioides proteinantigens or toxoids, such as tetanus toxoid, diphtheria toxoid, choleratoxoid, ovalbumin, or keyhole limpet haemocyanin).

In another embodiment, the present invention provides polynucleotidebased vaccines or immune-stimulatory formulations, whereby thepolynucleotide(s) encoding the polypeptides are administered directly tothe subject patient in need thereof, provided the polynucleotide has theappropriate transcriptional control regions to direct the expression ofthe coding sequence contained in the polynucleotide or expressionvector.

Therefore, the present invention also provides DNA based vaccines orimmunogenic compositions to provide one or more of the polypeptidesdescribed herein. DNA vaccines have been developed for a number ofdiseases, whereby a DNA vaccine contains a DNA encoding an antigencloned in a plasmid vector. It will be apparent to one skilled in theart that the immunostimulatory activity of the polypeptides encoded bythe DNA sequences disclosed herein lies not in the precise nucleotidesequence of the DNA sequences, but rather in the epitopes inherent inthe amino acid sequences encoded by the DNA sequences. It will thereforealso be apparent that it is possible to recreate the immunostimulatoryactivity of one of these polypeptides by recreating the epitope, withoutnecessarily recreating the exact DNA sequence. Such sequences may differby reason of the redundancy of the genetic code from the sequencesdisclosed herein. Accordingly, the degeneracy of the genetic codefurther widens the scope of the present invention as it enables majorvariations in the nucleotide sequence of a DNA molecule whilemaintaining the amino acid sequence of the encoded protein or a proteinthat consists essentially of the same sequence. Such degeneracy isdescribed in U.S. Pat. No. 6,228,371, the contents of which areincorporated herein by reference.

The expression of the antigen or polypeptide may be improved byproviding a strong promoter such as, for example, Rous Sarcoma VirusLTR, the cytomegalovirus immediate early promoter, and the SV40 Tantigen promoter. Success with DNA vaccines has been demonstrated usinga variety of antigens for a number of diseases (U.S. Pat. Nos.6,384,018, 6,284,533, 6,165,993, the contents of which are incorporatedherein by reference). The DNA vaccine or immune stimulating compositionmay further include an acceptable carrier or liposome as described inU.S. Pat. No. 5,703,055, which is incorporated herein by reference; andcan be made in accordance with known methods as described in, forexample, U.S. Pat. Nos. 5,589,466; 5,580,859; 5,561,064; and 6,339,068,the contents of which are incorporated herein by reference.

The delivery of the DNA vaccine or immunostimulatory composition can beaccomplished using a variety of procedures commonly employed in the art.For non-viral DNA transfer in cultured cells, examples of such methodsinclude-calcium phosphate mediated, DEAE-dextran, electroporation,direct microinjection, liposome mediated delivery, cell sonication, andreceptor mediated gene targeting which utilize a cell-receptor-specificligand and an DNA binding agent, which mediate the uptake of a gene intoa specific cell type based on the interaction of the ligand and thereceptor. The recombinant DNA encoding the polypeptides of the presentinvention can also be provided to the cells by direct injection of thenaked DNA or plasmid DNA or coupled to particle bombardment with knownmethods as described in, for example, U.S. Pat. No. 5,865,796,incorporated herein by reference.

In another embodied method of delivering the DNA vaccine orimmunostimulatory composition to the cell, viral-vector mediateddelivery can be used. Examples of viral vectors for such deliveryinclude adeno-associated virus (AAV) (U.S. Pat. No. 5,843,742incorporated herein by reference), adenovirus (U.S. Pat. Nos. 6,410,010and 6,403,370, incorporated herein by reference), vaccinia virus (U.S.Pat. Nos. 6,287,570, and 6,214,353 incorporated herein by reference),herpesvirus, canarypox virus (U.S. Pat. No. 6,183,750 incorporatedherein by reference), other Poxviruses, Retrovirus, and other RNA or DNAviral expression vectors known in the art.

The vectors used to deliver the polypeptides of the present inventionmay be maintained as an episome or stably integrated into the chromosomeof the cell.

IV. How the Polypeptide may be Isolated.

The peptides and polypeptides of the present invention, when produced,can be purified by isolation and purification methods for proteinsgenerally known in the field of protein chemistry. Within the context ofthe present invention, “isolated” means separated out of its naturalenvironment. An “isolated polypeptide” is, in this context, asubstantially pure polypeptide.

The term “substantially pure polypeptide” means a polypeptide that hasbeen separated from at least some of those components which naturallyaccompany it, such as other contaminating polypeptides, polynucleotides,and or other biological materials often found in cell extracts.Typically, the protein is substantially pure when it is at least 60%, byweight, free from the proteins and other naturally-occurring organicmolecules with which it is naturally associated in vivo. Preferably, thepurity of the preparation is at least 75%, more preferably at least 90%,and most preferably at least 99%, by weight. A substantially pureAg2/PRA polypeptide or polypeptide fragment may be obtained, forexample, by extraction from a natural source, or by expression of arecombinant nucleic acid encoding an immunoreactive Ag2/PRA1-106 orAg2/PRA27-106 polypeptide, such as the nucleic acid molecule shown asSEQ ID NO: 3 or SEQ ID NO:6, respectively, using methods describedherein. In addition, an amino acid sequence consisting of at least animmunogenic portion of the amino acid sequence of SEQ ID NO: 5 or SEQ IDNO:7 or SEQ ID NO:9 or SEQ ID NO:11 can be chemically synthesized in asubstantially pure form.

Methods of purification include, for example, extraction,recrystalization, ammonium sulfate precipitation, sodium sulfate,centrifugation, dialysis, ultrafiltration, adsorption chromatography,ion exchange chromatography, hydrophobic chromatography, normal phasechromatography, reversed-phase chromatography, gel filtration method,gel permeation chromatography, affinity chromatography, electrophoresis,countercurrent distribution, combinations of these, and other knowprotein or peptide purification methods are well known to those of skillin the art and can be used herein.

Purity can be measured by any appropriate method, e.g., HPLC analysis,immunoaffinity chromatography using an antibody specific for the Ag2/PRApolypeptide fragment, polyacrylamide gel electrophoresis, and the like.

A Coccidioides spp. polypeptide that is “isolated to homogeneity,” asapplied to the present invention, means that the Coccidioides spp.polypeptide has a level of purity where the Coccidioides spp.polypeptide is substantially free from other proteins, peptides andbiological components. For example, an isolated Coccidioides spp.polypeptide will often be sufficiently free of other peptide and proteincomponents so that sequencing may be performed successfully or thatpharmaceutically acceptable formulations can be created. However, thisdoes not exclude the re-mixing of the peptides of the invention, onceisolated, with other vaccine components.

V. Preparation and Formulation of Vaccines.

The polypeptides and formulations employing the polypeptides may also bein the form of a peptide salt thereof. In view of the utility of thepolypeptides of the present invention, preferred salts include thosesalts that are pharmaceutically acceptable for administration into asubject patient.

The polypeptides of the present invention may form a salt by addition ofan acid. Examples of the acid include inorganic acids (such ashydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, andsulfuric acid) or organic carboxylic acids (such as acetic acid,propionic acid, maleic acid, succinic acid, malic acid, citric acid,tartaric acid, and salicylic acid), acidic sugars such as glucuronicacid, galacturonic acid, gluconic acid, ascorbic acid, etc., acidicpolysaccharides such as hyaluronic acid, chondroitin sulfates, alginicacid, or organic sulfonic acids (such as methanesulfonic acid, andp-toluenesulfonic acid), etc.

The polypeptides of the present invention may also form a salt with abasic substance. Examples of these basic salts include, for example,salts with inorganic bases such as alkali metal salts (sodium salt,lithium salt, potassium salt, etc.), alkaline earth metal salts,ammonium salts, and the like or salts with organic bases, such asdiethanolamine salts, cyclohexylamine salts, and the like.

In one embodiment of the present invention, the various polypeptides ofthe present invention may be admixed in various combinations and oradmixed with other known proteins or peptides, which are known orbelieved to facilitate an immunological response, thereby providingprotection against Coccidioides spp. infection. In an alternativeembodiment, the polypeptides of the present invention may beadministered separately, i.e., at different time points, from each orfrom other proteins or peptides, which are known or believed tofacilitate an immunological response, thereby providing protectionagainst Coccidioides spp. infection. For example, the peptide of aminoacids 37 to 142 of SEQ ID NO:5 can be combined with one or moreadditional Coccidioides spp. polypeptides or antigens, such as Csa,Gel1, Ure, or non-Coccidioides protein antigens or toxoids, such astetanus toxoid, diphtheria toxoid, cholera toxoid, ovalbumin (OVA), orkeyhole limpet haemocyanin (KLH).

The pharmaceutically acceptable carriers which can be used in thepresent invention include, but are not limited to, an excipient, astabilizer, a binder, a lubricant, a colorant, a disintegrant, a buffer,an isotonic agent, a preservative, an anesthetic, and the like which arecommonly used in a medical field.

Also, the dosage form, such as injectable preparations (solutions,suspensions, emulsions, solids to be dissolved when used, etc.),tablets, capsules, granules, powders, liquids, liposome inclusions,ointments, gels, external powders, sprays, inhalating powders, eyedrops, eye ointments, suppositories, pessaries, and the like, can beused appropriately depending on the administration method and thepolypeptides of the present invention can be accordingly formulated.Pharmaceutical formulations are generally known in the art and aredescribed, for example, in Chapter 25.2 of Comprehensive MedicinalChemistry, Volume 5, Editor Hansch et al, Pergamon Press 1990.

The present invention also provides compositions containing thepolypeptides or fragments thereof containing one or more suitableadjuvants commonly used in the field of immunology and medicine toenhance the immune response in a subject. Examples of such adjuvantsinclude monophosphoryl lipid A (MPL), a detoxified derivative of thelipopolysaccharide (LPS) moiety of Salmonella minnesota R595, which hasretained immunostimulatory activities and has been shown to promote Th1responses when co-administered with antigens (see U.S. Pat. No.4,877,611; Tomai et al., Journal of Biological Response Modifiers. 1987.6:99–107; Chen et al., Journal of Leukocyte Biology 1991. 49:416–422;Garg & Subbarao. Infection and Immunity. 1992. 60(6):2329–2336; Chase etal., Infection and Immunity.1986. 53(3):711–712; Masihi et al, Journalof Biological Response Modifiers. 1988. 7:535–539; Fitzgerald, Vaccine1991. 9:265–272; Bennett et al, Journal of Biological Response Modifiers1988. 7:65–76; Kovach et al., Journal of Experimental Medicine, 1990.172:77–84; Elliott et al., Journal of Immunology. 1991.10:69–74; WheelerA. W., Marshall J. S., Ulrich J. T., International Archives of Allergyand Immunology October 2001;126(2):135–9; and Odean et al., Infectionand Immunity 1990. 58(2):427–432); MPL derivatives (see U.S. Pat. No.4,987,237) other general adjuvants (see U.S. Pat. No. 4,877,611); CpGand ISS oligodeoxynucleotides (see U.S. Pat. No. 6,194,388; U.S. Pat.No. 6,207,646; U.S. Pat. No. 6,239,116; U.S. Pat. No. 6,339,068;McCluskie, M. J., and H. L. Davis. Vaccine 2002. 19:413–422; Ronaghy A,Prakken B J, Takabayashi K, Firestein G S, Boyle D, Zvailfler N J, RoordS T, Albani S, Carson D A, Raz E. Immunostimulatory DNA sequencesinfluence the course of adjuvant arthritis. Journal of Immunology 2002.168(1):51–6.; Miconnet et al (2002) 168(3) Journal of Immunology pp1212–1218; Li et al (2001) Vaccine 20(1–2):148–157; Davis (2000)Devopmental Biology 104:165–169; Derek T. O'Hagan, Mary Lee MacKichan,Manmohan Singh, Recent developments in adjuvants for vaccines againstinfectious diseases, Biomolecular Engineering 18 (3) (2001) pp. 69–85;McCluskie et al (2001) Critical Reviews in Immunology 21(1–3):103–120);trehalose dimycolate (see U.S. Pat. No. 4,579,945); amphipathic andsurface active agents, e.g., saponin and derivatives such as QS21 (seeU.S. Pat. No. 5,583,112); oligonucleotides (Yamamoto et al, JapaneseJournal of Cancer Research, 79:866–873, 1988); detoxified endotoxins(see U.S. Pat. No. 4,866,034); detoxified endotoxins combined with otheradjuvants (see U.S. Pat. No. 4,435,386); combinations with QS-21 (seeU.S. Pat. No. 6,146,632); combinations of detoxified endotoxins withtrehalose dimycolate and endotoxic glycolipids (see U.S. Pat. No.4,505,899); combinations of detoxified endotoxins with cell wallskeleton (CWS) or CWS and trehalose dimycolate (see U.S. Pat. Nos.4,436,727, 4,436,728 and 4,505,900); combinations of just CWS andtrehalose dimycolate, without detoxified endotoxins (as described inU.S. Pat. No. 4,520,019); chitosan adjuvants (see U.S. Pat. Nos.5,912,000; 5,965,144; 5,980,912; Seferian, P. G., and Martinez, M. L.Immune stimulating activity of two new chitosan containing adjuvantformulations (2001) Vaccine. 2000. 19(6):661–8). All of the referencescited in this paragraph are incorporated herein by reference.

In another embodiment, the antigenic compositions of the presentinvention can be provided as an adsorbed vaccine or immunostimulatorycomposition as described in Matheis et al. (Matheis, M., Zott, A.,Schwanig, M. 2000. The role of the adsorption process for production andcontrol combined adsorbed vaccines. Vaccine 20:67–73), which isincorporated herein by reference.

In another embodiment, various adjuvants, even those that are notcommonly used in humans, may be employed in animals where, for example,one desires to subsequently obtain activated T cells or to protectvaluable or valued animals from infection due to Coccidioides spp.

VI. Administration of Vaccines

As used herein the subject that would benefit from the administration ofthe polypeptide and or nucleotide vaccines and formulations describedherein include any mammal which can benefit from protection againstCoccidioides spp. infection. In a preferred embodiment, the subject is ahuman. In a second embodiment, the subject is a domestic animal,including but not limited to dog, cat, horse, bovine (meaning any sex orvariety of cattle) or other such domestic animals.

By polypeptides capable of eliciting an immune response in a subjecthuman, including vaccination, the invention covers any polypeptide,peptide, peptide mimic, or chemical product capable of inducing animmune reaction that results in or augments the subject's ability tomount some level of immune protection inhibiting Coccidioides spp.infection. In one embodiment, the Coccidioides spp. is Coccidioidesimmitis. In another embodiment, the Coccidioides spp. is Coccidioidesposadasii.

As used herein, “inhibit”, “inhibiting” or “inhibition” includes anymeasurable or reproducible reduction in the infectivity of Coccidioidesspp. in the subject patient. “Reduction in infectivity” means theability of the subject to prevent or limit the spread of Coccidioidesspp. fungus in tissues or organs exposed or infected by said fungus.Furthermore, “amelioration”, “protection”, “prevention” and “treatment”mean any measurable or reproducible reduction, prevention, or removal ofany of the symptoms associated with Coccidioides spp. infectivity, andparticularly, the prevention, or amelioration of Coccidioides spp.infection and resultant pathology itself.

The dosages used in the present invention to provide immunostimulationinclude from about 0.1 μg to about 500 μg, which includes, 0.5, 1.0,1.5, 2.0, 5.0, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, and 450 μg, inclusiveof all ranges and subranges there between. Such amount may beadministered as a single dosage or may be administered according to aregimen, including subsequent booster doses, whereby it is effective;e.g., the compositions of the present invention can be administered onetime or serially over the course of a period of days, weeks, months andor years.

The polypeptide compositions of the present invention can beadministered by any suitable administration method including, but notlimited to, injections (subcutaneous, intramuscular, intracutaneous,intravenous, intraperitoneal), eye dropping, instillation, percutaneousadministration, transdermal administration, oral administration,intranasal administration, inhalation, etc.

VII. Other Uses.

Also included within the scope of the present invention are kitssuitable for providing one or more of the polypeptides of the invention.For example, in such a kit one vial can comprise the polypeptides of theinvention admixed with a pharmaceutically acceptable carrier, either ina aqueous, non-aqueous, or dry state; and a second vial which can carryimmunostimulatory agents, and or a suitable diluent for the peptidecomposition, which will provide the user with the appropriateconcentration of peptide to be delivered to the subject. In oneembodiment, the kit will contain instructions for using the polypeptidecomposition and other components, as included; such instructions can bein the form of printed, electronic, visual, and or audio instructions.The vaccinations will normally be at from two to twelve week intervals,more usually from three to five week intervals. Periodic boosters atintervals of 1–5 years, usually three years, will be desirable tomaintain protective levels. The course of the immunization may befollowed by assays for activated T cells produced, skin-test reactivity,or other indicators of an immune response to Coccidioides spp.

The polypeptide of the invention can be used to detect the presence ofantibodies in the sera of patients potentially infected withCoccidioides spp. Antibodies that react specifically with the inventivepolypeptides can be used to detect the presence of circulating antigensin the sera of patients potentially infected with Coccidioides spp. Suchdetection systems include radioimmunoassays and various modificationsthereof which are well-know to those skilled in the art. In addition,the polypeptide of the invention can be used to detect the presence of acell-mediated immune response in a biological sample. Such assay systemsare also well-known to those skilled in the art and generally involvethe clonal expansion of a sub-population of T cells or the production ofcytokines in response to stimuli from the polypeptide or detection ofreactive T cells by flow cytometry or other methods known to thoseskilled in the art; e.g., methods described by Richards et al.(Richards, J. O., Ampel, N. M., Galgiani, J. N. and Lake, D. F. 2001.Dendritic cells matured by Coccidioides immitis lysate induce antigenspecific naive T cell activation. Journal of Infectious Diseases184:1220–1224). When so-used, the humoral and or cell-mediated responseof a patient can be determined and monitored over the course of thedisease. Methods of generating antibodies directed to a specific peptidefragment are known in the art. Examples of such methods are disclosed inAntibodies, A Laboratory Manual, Harlow and Lane, Cold Spring HarborPress, 1988, herein incorporated by reference.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples that are providedherein for purposes of illustration only, and are not intended to belimiting unless otherwise specified.

EXAMPLES Example 1

Immunoprotection Studies with Recombinant Ag2/PRA Vaccines Expressed inE. coli

Materials and Methods

Cloning, Expression, and Characterization of Recombinant Vaccines Designof Ag2/PRA subunits. Potential antigenic domains were identified in thepredicted 194 amino acid sequence of Ag2/PRA (Dugger, Kris Orsborn, KaraM. Villareal, An Ngyuen, Charles R. Zimmermann, John H. Law and John N.Galgiani. 1996. Cloning and Sequence Analysis of the cDNA for a Proteinfrom Coccidioides immitis with Immunogenic Potential. BiochemicalBiophysical Research Communication 218:485–489; Genbank Accession numberU39835). We used the antigenicity prediction algorithm of thePEPTIDESTRUCTURE program of the GCG Package (Genetics Computer Group,Madison, Mich. [now Accelrys, San Diego, Calif.]), which analyzes sixproperties relating to secondary peptide structure and calculates the“antigenic index” as defined for antibody based immune responses tomodel proteins, and the ANTIGEN program of PC Gene (Intelligenetics,subsumed by Accelrys), which analyzes the hydrophilicity profile ofpolypeptides. Sequences were examined for breakpoints betweenhigh-scoring fields which were identified by both programs, and theseboundaries were used as a guide in designing four overlapping subunits,corresponding to amino acids 1-40, 27-106, 90-150 and 125-194. In orderto not miss epitopes, sequences were also created in which thefull-length protein was divided approximately in half, separating theN-terminal cysteine motifs and C-terminal proline-threonine-rich and GPIanchor motifs, with a 17-amino acid overlap (1-106 and 90-194). Lastly,a sequence was created in which the cysteine motif was separated fromthe signal peptide (19-100).

Construction of plasmids. Total RNA was extracted from 48-h spherules ofC. posadasii strain Silveira and reverse transcribed with Superscript IIand oligo(dT) as previously described (Duger, K. O., Villareal, K. M.,Ngyuen, A., Zimmermann, C. R., Law, J. H., and Galgiani, J. N. 1991. Animmunoreactive apoglycoprotein purified from Coccidioides immitis.Infection and Immunity 59:2245–2251). The resulting cDNA was used as atemplate in a 3-step PCR process catalyzed by Pfu DNA polymerase(Stratagene, San Diego, Calif.) for 35 cycles. PCR reactions consistedof 1×Pfu buffer (10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-Cl pH 8.75, 2 mMMgSO₄, 0.1% Triton X-100 and 0.1 mg/ml BSA), 2 μl template, 0.4 mMdNTP's, and 5U Pfu polymerase. The nucleotide sequences of the sense andantisense PCR primers were 5′ CCGGATCCATGCAGTTCTCTCACGCTC 3′(P1, SEQ IDNO:12) and 5′CCGAATTCAGTGAAATCAGGTGTGTT 3′ (P2, SEQ ID NO:13), each ofwhich includes a restriction site to facilitate subcloning (see Tables 1and 2). The resulting 970 bp product encoding the full-length Ag2/PRAwas gel purified, digested with BamHI and EcoRI, and ligated into theBamHI/EcoRI sites of pBluescript SK⁺ (Stratagene) by standard techniquesto create pCiAg33.41 ( J. Sambrook, E. F. Fritsch and T. Maniatis. 1989.Molecular Cloning: A Laboratory Manual, 2^(nd) ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.).

The sequence of the cDNA insert was confirmed by automated DNAsequencing on an ABI 377 sequencer (Macromolecular Structures Facility,Arizona Research Labs, University of Arizona). The insert was excisedfrom pBluescript with BamHI and EcoRI, and ligated into pET32a (Novagen,Madison, Wis.) to produce pPRA.B15. The orientation and frame wereconfirmed by sequencing, and the plasmid transformed into E. coliBL21(DE3)SlyD⁻ (kind gift of Ry Young, Texas A&M University) by a CaCl₂method as described (Theo N. Kirkland, Fred Finley, Kris I. Orsborn andJohn N. Galgiani. 1998. Evaluation of the Proline-Rich Antigen ofCoccidioides immitis as a Vaccine Candidate in Mice. Infection andImmunity 66:3519–3522; and J. Sambrook, E. F. Fritsch and T. Maniatis.1989. Molecular Cloning:. A Laboratory Manual, 2^(nd) ed. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.). To create plasmidsencoding the peptide subunits of Ag2/PRA, a series of oligonucleotidePCR primers corresponding to the desired sequences, includingrestriction sites for BamHI and EcoRI were designed (see Tables 1 and2). Stop codons were included on antisense primers as appropriate.Primer pairs were used in a set of 2-step PCR reactions with pPRA.B 15as template and Pfu DNA polymerase as above and the resulting amplimerscloned into the BamHI/EcoRI sites of pET28a (Novagen) or pET32a bystandard methods (see Table 2) for expression (see Table 3).

TABLE 1 Primers used in cloning/PCR procedures Primer No. SEQ ID NO:Primer sequence* Restiction Site Added P1 12CCGGATCC-ATGCAGTTCTCTCACGCTC BamHI P2 13 CCGAATTC-AGTGAAATCAGGTGTGTTEcoRI P3 16 CCGGATCC-GCTCTCAACTGCTTCGTTG BamHI P4 15 GGGAATTCTTA- EcoRI,stop GGTCTCGGATGGCTCGGG P5 29 CCGGATCC- BamHI ATTGACATCCCACCAGTTG P6 30CCGAATTCTTA- EcoRI, stop GCCAGTGGGGACACCACC P7 31CCGAATTC-GTGCTTGTCAGTTTTGCT EcoRI P8 17 GGAGATCTTTA- BglII, stopGGTCTCGGATGGCTCGGG P9 32 CCAGATCTTTA- BglII, stop GCCAGTGGGGACACCACC P1033 CCCCAGATC-GTGCTTGTCAGTTTTCGT BglII P11 19CCCGTCGACTTA-GGTCTCGGATGGCTC SalI, stop P12 34CCGGATCC-CAGCTCCCAGACATCCCA BamHI P13 35 CGAATTCTTA- EcoRI, stopAGCGGCGGTGGTGTCAAC P14 25 CCGAATTC-ATGCAGTTCTCTCACGC EcoRI P15 26TTGGATCC-GGTCTCGGATGGCTCG BamHI P16 27 CCCGGATCC-ATGAAGTTCTCACTCCTCBamHI P17 28 CCCGTCGACTTA-TTTCAACCCGCAC SalI, stop *hyphen indicatesinterface of cDNA homology sequence and nucleotides for restrictionsites, stop codons, and clamps

TABLE 2 Summary information for cloning and expression Expressedpolypeptide/ Encoded vaccine (plasmid Primers Template Recipientdesignation) used Used vector Host Ag2/PRA 1–194 P1/P2 cDNA pET32a E.coli (pPRA.B 15) Ag2/PRA 27–106 P3/P4 pCiAg33.41 pET28a E. coli Ag2/PRA90–151 P5/P6 pCiAg33.41 pET28a E. coli Ag2/PRA 1–106 P1/P4 pPRA.B15pET32a E. coli Ag2/PRA 90–194 P5/P7 pPRA.B15 pET28a E. coli Ag2/PRA19–100 P12/P13 pCVP20.17 pET32a E. coli Ag2/PRA 1–194 P1/P10 pCiAg33.41pVR1020 Mammalian (pCVP20. 17) host Ag2/PRA 1–106 P1/P8 pCiAg33.41pVR1020 Mammalian (pTPA) host Ag2/PRA 90–194 P6/P10 pCiAg33.41 pVR1020Mammalian (pTPB) host Ag2/PRA 27–106 P5/P8 pCiAg33.41 pVR1020 Mammalian(pTP2) host Ag2/PRA 90–151 P6/P9 PCiAg33.41 pVR1020 Mammalian (pTP3)host Ag2/PRA 1–106 P1/P11 PCVP20.17 YEpFLAG-1 S. cerevisiae Ag2/PRA1–106 P14/P15 PCVP20.17 YEpFLAG-1 S. cerevisiae (Ag2/PRA 1–106 + Csa)Csa P16/P17 PET28b- YEpFLAG-1 S. cerevisiae (Ag2/PRA CSA 1–106 + Csa)*full length cDNA clone

Construction of plasmid vaccines. A transcript encoding Ag2/PRA (aminoacids 1-194) was cloned into the mammalian expression vector VR1020(Vical, Inc., San Diego, Calif.). to create pCVP20.17. Briefly, the fulllength cDNA clone, pCiAg33.41 (Dugger et.al.1996), was used as atemplate with primers P1 and P2 (see Tables 1 and 2) in a two-step PCRcatalyzed by Pfu DNA polymerase (Stratagene), and the resulting 615 bpamplimer ligated into the BamHI/BglII sites on pVR1020 by standardtechniques to create pCVP20.17 (Abuodeh, R. O., Shubitz, L. F., Siegel,E., Snyder, S., Peng, T., Orsborn, K. I., Brummer, E., Stevens, D. A.,and Galgiani, J. N. 1999. Resistance to Coccidioides immitis in miceafter immunization with recombinant protein or DNA vaccine of aproline-rich antigen. Infection and Immunity 67:2935–2940). To makeoverlapping subunits in the same vector, oligonucleotide primerscorresponding to the desired sequence (see FIG. 3) plus restrictionsites for BamHI or BglII (see Tables 1 and 2) were used in to createsequences encoding the desired peptide fragments. The amplifiedfragments were cloned into the BamHI/BglII sites of VR1020 to createplasmids pTP-A (corresponding to Ag2/PRA1-106), pTP-B (corresponding toAg2/PRA 90-194), pTP-Super2 (corresponding to Ag2/PRA19-100), pTP-2(corresponding to Ag2/PRA 27-106) and pTP-3 (corresponding to Ag2/PRA90-151) using the previously described methods. The orientation, frameand sequence of plasmid inserts were confirmed by DNA sequencing. DNAfor use as a vaccine was prepared with the Qiagien Gigaprep kit(Chatsworth, Calif.), ethanol precipitated, and adjusted to aconcentration of 1 mg/ml in phosphate buffered sterile saline and storedat −20° C. until use.

Expression and purification of recombinant peptides. Procedures foroptimal expression rAg2/PRA and its subunits were developed andconditions varied individually as shown in Table 3. Growth and inductionwas essentially as described (Kirkland et al. 1998), with changes inmedium composition and IPTG concentration as noted, except that mediumfor initial overnight growth of the host cells contained 10% glucose toinhibit premature expression of the polypeptides.

TABLE 3 Conditions for expression of recombinant Ag2/PRA and itssubunits. Sequences* Vector Competent Cell Medium IPTG (mM)  1–194PET32a BL21(DE3)slyD- SB 0.5  1–106 PET32a BL21(DE3) TB 0.25 19–100PET32a BL21(DE3) LB 0.5 90–194 PET28a Tuner(DE3)plysS TB 0.5 27–106PET28a BL21(DE3)star LB 0.5 *refers to corresponding amino acid numbersof native Ag2/PRA sequence

pET32 and pET28 both encode a 6×-his tag, which facilitates purificationby immobilized metal affinity chromatography (IMAC). Full length Ag2/PRAwas purified by batch IMAC under denaturing conditions onNi-nitrilotriacetic acid (NTA) agarose (Qiagen, Inc., Chatsworth,Calif.) by elution with a pH step gradient essentially as described inKirkland et. al. 1998, with the following changes. Column bufferconsisted of 8M urea, 100 mM NaH2PO4, 10 mM Tris, pH 8.0 and 10%glycerol. Renaturation buffer consisted of 6.5M urea, 150 mM NaCl, 1 mMEDTA, 5 mM glutathione, 0.5 mM GSSH (oxidized glutathione), 20 mM Tris,pH 9.5 and 10% glycerol. The urea concentration of renaturation bufferwas stepped down every 24 hours to 5M, 3.5M, 1M and finally 0 M urea.

Polypeptides expressed in pET32a (Ag2/PRA 1-194 and Ag2/PRA 1-106,sequence ID 4) were subjected to thrombin cleavage, to remove somevector encoded amino acids prior to final purification and use asvaccines. The fusion polypeptides were dialyzed into thrombin cleavagebuffer, 20 mM Tris, pH 8.0, 150 mM NaCl and the pH adjusted to pH 8.4.CaCl₂ (to 2.5 mM) was added and the 6×-his tag removed by digestion withbiotinylated thrombin as described (Kirkland et. al. 1998). Afterseparation from the 6×-his tag, the protein was dialyzed intophysiological buffer consisting of 5.3 mM KCl, 0.4 mM KH2PO4, 137 mMNaCl, 0.3 mM Na2HPO4 and 10 mM DTT, concentrated and quantitated.

Polypeptides expressed in pET28a (see Table 2) were not subjected tothrombin cleavage. Following step down urea dialysis, these polypeptideswere dialyzed directly into physiological buffer, employing dialysistubing and spin concentrator units with smaller molecular weightcutoffs.

The recombinant Ag2/PRA1-106 fusion polypeptide of the present inventionexpressed by the transformed bacterium was 271 amino acids in length andcontains 165 amino acids encoded by the pET32a vector (SEQ ID NO:4). 130amino acids, including the 6×-his tag, are removed by thrombin cleavage,leaving 36 vector-encoded and 106 Ag2/PRA1-106-encoded amino acids onthe vaccine polypeptide (SEQ ID NO: 5). The recombinant Ag2/PRA27-106fusion polypeptide of the present invention expressed by the transformedbacterium is 114 amino acids in length and contains 34 amino acidsencoded by the pET28a vector and retains the 6×-his tag (SEQ ID NO:7).

Polypeptides were separated by SDS-PAGE on 12.5% polyacrylamide(Tris/glycine buffered) or 16.5% polyacrylamide (Tris/glycine buffered)gels (Bio-Rad, Hercules, Calif.), and the purity of recombinant proteinswas assessed as greater than 95% by Coomassie Blue staining. Immunoblotswere probed with a goat antiserum (Dugger et. al. 1996) tospherule-derived Ag2/PRA as previously described (Kirkland et al, 1998)or with anti-T7-Tag antibody conjugated to alkaline phosphatase(Novagen).

Immunization and Protection Studies

Mice. Female, 6-weeks old BALB/c mice were purchased fromHarlan-Sprague-Dawley (Indianapolis, Ind.) and maintained inconventional housing under microisolation lids. Mice were immunizedwithin one week of receipt. At the time of infection, mice were moved toBiosafety Level 3 housing where they remained for the duration of theirexperiment.

Immunizations. Immunization with various plasmid and recombinant peptidevaccines was carried out as previously described with groups of eight ormore mice (Abuodeh R O, Shubitz L F, Siegel E, Snyder S, Peng T, OrsbornK I, Brummer E, Stevens D A, Galgiani J N. 1999 Resistance toCoccidioides immitis in mice after immunization with recombinant proteinor DNA vaccine of a proline-rich antigen. Infection and Immunity67:2935–40.). For DNA vaccination, mice were immunized twice 4 weeksapart with 50 μl of plasmid in various concentrations as indicated inthe results injected into each cranial tibial muscle. Unimmunizedcontrol mice received the vector without insert (VR1020). Forvaccination with recombinant peptides, protein suspended in 0.9% sterilesaline was combined with MPL-SE adjuvant (Corixa, Inc, Hamilton, Mont.)according to the manufacturer's instructions. Mice were vaccinated twice4 weeks apart subcutaneously in the inguinal region with 1 μg of proteinper dose.

Infection. Mice were infected intraperitoneally one month after boosterimmunizations as previously described (Abuodeh et al., 1999), usingarthroconidia of C. immitis, strain RS, were kindly supplied by Dr. TheoKirkland (University of California, San Diego). For each study,viability and the size of the infecting inoculum was confirmed by platecount at the time of infection (Abuodeh et al. 1999).

Fourteen days after infection, all mice were sacrificed with an overdoseof inhalant anesthesia and the right lung removed aseptically. Organswere homogenized, diluted, and plated. Colony-forming units (CFU) wereenumerated at three days and reported as Log₁₀ CFU/organ.

Statistical analyses. For fungal burden of organs of infected animals,comparisons between groups were determined by analysis of variance(ANOVA) and Duncan's multiple range tests as implemented by SAS (SASInstitute, Inc., Cary, N.C., software version 6.12). The type Iexperiment-wise error rate of 0.05 was used to control for multiplecomparisons.

Results

Differential effects of DNA subunit vaccines. In addition to pCVP20.17,which encodes the full-length 194-amino acid Ag2/PRA, plasmid vaccineswere constructed with inserts encoding amino acid sequences 1-106,27-106, 90-151, and 90-194 of Ag2/PRA. In two separate studies,vaccination with each of these plasmids was compared to the plasmidvector alone, using doses and conditions based on previous studies withDNA vaccines encoding full-length Ag2/PRA. As shown in Table 4,significantly fewer colonies of Coccidioides spp. were isolated fromlungs of mice vaccinated with plasmids encoding either Ag2/PRA1-106 orAg2/PRA27-106 than from mice receiving the vector alone. Furthermore,the plasmid encoding Ag2/PRA27-194 was less effective than the plasmidencoding full-length Ag2/PRA. In contrast, there was virtually nobeneficial effect of vaccination with plasmids encoding eitherAg2/PRA90-151 or Ag2/PRA90-194.

TABLE 4 Fungal burdens in lungs of mice challenged with Coccidicidesspp. Geometric Mean CFU* Plasmid Vaccine Group Exp. 1 Exp. 2 VectorControl 4.92 4.20 rAg2/PRA 90–194 N.D. 4.48 rAg2/PRA 90–151 4.80 4.05rAg2/PRA 27–106 2.90 3.15 rAg2/PRA 1–106 2.61 3.09 rAg2/PRA (fulllength) 2.01 1.94 *expressed as log₁₀ values N.D.: not determined

Protection afforded by recombinant peptide subunit vaccines. To comparetheir protective efficacy as vaccines in mice, recombinant Ag2/PRA,rAg2/PRA1-106, rAg2/PRA27-106, and rAg2/PRA90-194 were expressed in E.coli, the polypeptides were isolated and used to immunize mice that werethen challenged with C. immitis in a fungal burden assay. The resultsfrom quantitative cultures of the target organ lungs are shown in Table5. As previously seen with plasmid vaccination data described above,mice vaccinated with full-length rAg2/PRA or either rAg2/PRA1-106 orrAg2/PRA27-106 of the present invention were significantly protected ascompared to mice receiving adjuvant alone. Also similar to the previousstudy, vaccination with rAg2/PRA1-106

TABLE 5 Fungal burdens in lungs of mice challenged with Coccidicidesspp. Group Geometric Mean CFU* Adjuvant Control 5.85 rAg2/PRA 90–1945.69 rAg2/PRA 90–151 2.58 rAg2/PRA 27–106 + 90–194 3.84 rAg2/PRA 27–1064.25 rAg2/PRA 1–106 + 90–194 2.58 rAg2/PRA 1–106 2.60 rAg2/PRA (fulllength) 2.91 *expressed as log₁₀ valueswas more protective under the conditions of the experiment than thatobtained by immunization with rAg2/PRA27-106. In contrast, vaccinationwith rAg2/PRA90-194 showed virtually no protection and the fungalburdens were similar to those seen in the adjuvant control. Furthermore,immunization by co-administration of rAg2/PRA90-194 with eitherrAg2/PRA106 or rAg2/PRA27-106 did not enhance the protection obtained byimmunization with the single, later recombinant polypeptides. Thesestudies with recombinant polypeptide vaccines fully corroborate thedetermination of the protective antigen domain of the N-terminal 1-106portion of the Ag2/PRA identified in the previous studies with subunitDNA vaccinations and provide strong support that the region of aminoacids 90-194 does not provide any additional enhancement over thatafforded by MPL adjuvant alone.

Example 2

Protection Studies with S. cerevisiae Expressed Ag2/PRA1-106 PolypeptideAntigen Used Singly or in Combination with a Second Coccidioides spp.Antigen

Materials and Methods

Cloning and Expression of Ag2/PRA1-106 in Saccharomyces cerevisiae

pCVP20.17 was used as a template in a PCR catalyzed by Pfu DNApolymerase for 35 cycles as described in Example 1 above. The senseprimer was P1 (5′CCGGATCCATGCAGTTCTCTCACGCT 3′)(Table 1 and SEQ IDNO:12) and the antisense primer P2 (5′ CCCGTCGACTTAGGTCTCGGATGGCTC3′)(SEQ ID NO:13), each of which includes a restriction site tofacilitate subcloning. The resulting 338 bp product encoding theAg2/PRA1-106 was purified using NucleoSpin Kit (Clontech, Palo Alto,Calif.), digested with BamHI and SalI, and ligated into the BamHI/SalIsites of the 7190 bp yeast expression vector YEpFLAG-1 (Sigma, St.Louis, Mo.) using the manufacturer's protocols. TheYEpFLAG-1-Ag2/PRA1-106 construct was used to transform E. coli DH5α bystandard techniques, and clones were screened and confirmed bysequencing as described in Example 1. The YEpFLAG-1 construct was thentransformed into the S. cerevisiae BJ3505 (Sigma, St. Louis, Mo.) hostusing a Yeast Transformation Kit (Sigma) by the manufacturer'sprotocols. Transformed yeast cells were selected by plating on SyntheticComplete Medium (SCM) minus tryptophan (Sigma).

The YEpFLAG-1-Ag2/PRA1-106+CSA chimeric construct was created using thestandard methods described above. The sense and antisense primers usedfor the creation of the chimeric construct comprise the sequences of SEQID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO: 28. The chimericYEpFLAG-1 construct was then transformed into the S. cerevisiae BJ3505(Sigma, St. Louis, Mo.) host using a Yeast Transformation Kit (Sigma) bythe manufacturer's protocols. Transformed yeast cells were selected byplating on Synthetic Complete Medium (SCM) minus tryptophan (Sigma).

Recombinant protein was produced by inoculating a yeast colony from theselective agar into 30 ml of SCM minus tryptophan and growing the cellsfor 48–72 hours at 30° C. until the cell density reached A₆₀₀=0.6.Twenty five ml of the starter culture was added to 500 ml of YP HighStability Expression Media (Sigma) containing 1% yeast extract, 8%peptone, 1% glucose, 3% glycerol and 20 mM CaCl₂, and the expressionculture was grown at 30° C. at 175 rpm for 96 hours. The culturesupernatant containing the rAg2/PRA1-106 fusion polypeptide or thechimeric Ag2/PRA1-106+Csa fusion protein, respectively, were separatedfrom yeast cells by centrifugation at 3000 rpm for 15 minutes.

The yeast-derived rAg2/PRA1-106 fusion polypeptide (SEQ ID NO:9) or thechimeric Ag2/PRA1-106+Csa fusion protein (SEQ ID NO:11), respectively,were purified by binding to anti-Flag-M1 Affinity Gel (Sigma) andelution with 0.1M glycine per the manufacturer's protocol. The fusionpolypeptides were collected and dialyzed in physiological buffer at 4°C. and stored at −70° C. until used. Protein concentration was measuredwith the BCA Protein Assay Kit (Pierce, Rockford Ill.) and the purityassessed by Coomassie stain of SDS-PAGE gels and immunoblot using ananti-Ag2/PRA goat antibody as described above. Purity was furtherassessed by mass spectrometry, using Bruker Reflex-III MALDI/TOF (BrukerDaltonics, Billerica, Mass.). The data were analyzed with software “Mover Z” (Genomic Solutions, Canada).

Mouse Immunization and Challenge Methods

Female, 6-weeks old C57B1/6 mice were purchased fromHarlan-Sprague-Dawley (Indianapolis, Ind.) and maintained inconventional housing under microisolation lids. Mice were divided intofour groups of 10 mice each for experimental testing of the recombinantpolypeptide antigens rAg2/PRA1-106, rCsa (derived and expressed byrecombinant methods from C. posadasii [encoded by the nucleotidesequence of SEQ ID NO:23; isolated by Dr. Garry Cole, Medical College ofOhio, Toledo, Ohio]), rAg2/PRA1-106+rCsa, or adjuvant control. Forvaccination with recombinant peptides, protein was suspended in 0.9%sterile saline and combined with MPL-SE adjuvant (Corixa) and CpG, animmunostimulatory oligonucleotide sequence purchased from Integrated DNATechnologies, Inc. The CpG ODN sequence used to immunize mice wasTCCATGACGTTCCTGACGTT (SEQ ID NO:24) (CpG motifs are underlined). Micewere vaccinated twice, 14 days apart, subcutaneously in the inguinalregion with 200 μl saline containing 1 μg of protein, 10 μg of CpG, andMPL-SE according to manufacturer's instructions. Controls received 10 μgCpG and MPL-SE. Animals were challenged intranasally 26 days after thelast administration of antigen with 50 arthroconidia of C. posadasiistrain Silveira. Animals were monitored for deaths for 56 days.Survivors were sacrificed with an overdose of inhalant anesthesia andthe right lung removed aseptically. Organs were homogenized, diluted,and plated on agar plates for the quantitative recovery of viableCoccidioides spp. Colony-forming units (CFU) were enumerated at threedays and reported as Log₁₀ CFU/organ.

Results

Mice began to look ill 14 days after challenge, with deaths ensuingthereafter. All mice in the adjuvant control group had died by Day 18post-infection. Animals were held for a total of 56 days and the in-lifeportion of the experiment was terminated. The survival results arepresented in Table 6. The data indicate pronounced survival in therAg2/PRA1-106 immunized mice in comparison to those immunized with rCsaor adjuvant control. What is also apparent is the enhanced protectionprovided by the combination of rAg2/PRA1-106 and rCsa in comparison tothe survival conferred by immunization with the single antigens alone,based both on percent survival at 56 days and mean days survived.

TABLE 6 Survival of mice challenged with C. posadasli Group % SurvivalMean Survival (Days) Adjuvant Control 0 16.2 rCsa 30 39.7 rAg2/PRA 1–10660 42.6 rAg2/PRA 1–106 + rCsa 90 52.2

The surviving mice were euthanized on Day 56 post-infection and thelungs were removed and processed for the recovery and enumeration of thefungal burden in this target organ. The results of the quantitativecultures are presented in Table 7. To normalize the results acrossexperimental groups, lungs from mice not surviving to Day 56 wereassigned a fungal burden value of 5.0×10⁶ cfu; a value that is twice thelevel possible to detect under the conditions of the experiment.

TABLE 7 Fungal burdens in lungs of mice challenged with Coccidicidesspp. Group Geometric Mean CFU* Adjuvant Control 6.70 rCsa 5.85 rAg2/PRA1–106 4.56 rAg2fPRA 1–106 + rCsa 3.92 *expressed as log₁₀ values

Closely reflecting the pattern observed among survival of the vaccinegroups, the fungal burdens of the mice immunized with the single antigenrAg2/PRA1-106 were reduced in comparison to those immunized with rCsaalone or in animals receiving the adjuvant control. The results of thefungal burden assay parallel those of the survival experiment in thatthe fungal burdens were further reduced in the group immunized by thecombination of rAg2/PRA1-106 and rCsa in comparison to the survivalconferred by immunization with the single antigens alone. The resultsthereby demonstrate the ability of the single recombinant proteinrAg2/PRA1-106 to serve as an effective immunizing vaccine, and that theprotection can be further enhanced by the addition of a secondCoccidioides spp. antigen; in this case rCsa. The data also provideevidence that the yeast-derived rAg2/PRA1-106 polypeptide produces aprotective immune response comparable to that seen with the comparableE. coli-derived polypeptide.

Example 3

Ex-vivo Production of Interferon-Gamma in Response to Single andCombination Antigen Vaccines

Materials and Methods

Female, 17-weeks old C57B1/6 mice were purchased fromHarlan-Sprague-Dawley (Indianapolis, Ind.) and maintained inconventional housing under microisolation lids. Mice were divided intogroups of 3 animals each for experimental testing of the recombinantpolypeptide antigens rAg2/PRA1-106 (yeast-derived), rCsa (obtained fromDr. Garry Cole), rAg2/PRA1-106+rCsa, or adjuvant controls. Forvaccination with recombinant peptides, the protein was suspended in 0.9%sterile saline and was combined with a mixture of 10 μg of CpG and 25 μgof MPL (Corixa, Inc., Hamilton, Mont.) adjuvants. Mice were vaccinatedtwice 14 days apart subcutaneously in the inguinal region with doses of1 μg of protein of each antigen or control adjuvants in a volume of 200μl. Thirty-seven days after the last immunization, mice were euthanizedand spleens were aseptically removed from animals, processed and placedin cell culture media as described previously [Abuodeh et al., 1999].Cells from each experimental group were stimulated with 10 μg/ml of eachof the following: bovine serum albumin (BSA) as negative control,Concanavalin A (Con A) as positive control, rAg/PRA1-106 polypeptide, orrCsa polypeptide. After stimulation for approximately 72 h, supernatantswere removed and assayed for the production of interferon gamma (IFN-γ),a key immunologic marker of a Th1 response, using an OptEIA kit(Pharmingen, Inc., San Diego, Calif.) according to the manufacturer'sprotocols.

Results

The results of the INF-γ assay are presented in Table 8. The positiveand negative controls provided IFN-γ responses consistent withexpectations. Stimulation of cells with the homologous antigens alsoresulted in marked responses, while stimulation with the heterologousantigens did not result in the release of detectable IFN-γ, a keyimmunologic marker of a Th1 response. The data indicate that vaccinationof mice with

TABLE 8 Production of IFN-γ from cells obtained from immunized mice andstimulated with recombinant C. posadasii polypeptide antigens ImmunizingIFN-γ(pg/ml) from Stimulus Antigen BSA Con A rAg2/PRA 1–106 rCsaAdjuvant Control ND*  6,228 ND ND rCsa NP  3,611 ND   652 rAg2/PRA 1–106ND 13,120  5,070 ND rAg2/PRA 1–106 + NP 20,000 22,419 1,496 rCsa *Notdetected: less than 125 pg/mlthe recombinant Coccidioides spp. polypeptides leads to an immunizedstate that can be detected by production of the cytokine IFN-γ inresponse to ex vivo stimulation of spleenic cells by the antigens. Therewas also a markedly enhanced response to stimulation by each of thesingle antigens in the cells from the mice immunized by the combinationof rAg2/PRA1-106+rCsa in comparison to the responses from mice immunizedwith the single antigens. The data suggest that immunization with thetwo C. posadasii antigens in combination may result in an additiveresponse, as measured by IFN-γ.

Obviously, numerous modifications and variations on the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

1. An isolated polypeptide encoded by the nucleotide sequence ofnucleotides 31 to 795 of SEQ ID NO:10, or an isolated polypeptidecomprising the amino acid sequence of amino acids 11 to 264 of SEQ IDNO:11.
 2. A composition comprising the isolated polypeptide of claim 1.3. The composition of claim 2, wherein said composition includes apharmaceutically acceptable carrier.
 4. The composition of claim 2,further including an adjuvant.
 5. The composition of claim 2, furthercomprising at least a second polypeptide.
 6. A method of generating animmune response in a mammal, comprising introducing into the mammal acomposition comprising the isolated polypeptide of claim 1, in an amountsufficient to elicit an immune response.
 7. The method of claim 6wherein said mammal is a human.
 8. The method of claim 6 wherein saidmammal is an animal selected from the group consisting of dog, cat,horse and bovine.
 9. A method of generating antibodies that recognizethe isolated polypeptide of claim 1 comprising introducing into a mammala composition comprising the isolated polypeptide of claim 1, in anamount sufficient to elicit an antibody response.
 10. The method ofclaim 9 wherein said mammal is a human.
 11. The method of claim 9wherein said mammal is an animal selected from the group consisting ofdog, cat, horse, goat, rat, mouse, rabbit and bovine.
 12. A method ofgenerating antibodies that recognize the isolated polypeptide of claim 1comprising introducing into a mammal a composition comprising theisolated polypeptide of claim 1 and a pharmaceutically acceptablecarrier, in an amount sufficient to elicit an antibody response.
 13. Amethod of generating antibodies that recognize the isolated polypeptideof claim 1 comprising introducing into a mammal a composition comprisingthe isolated polypeptide of claim 1 and an adjuvant, in an amountsufficient to elicit an antibody response.
 14. A kit, comprising theisolated polypeptide of claim
 1. 15. The kit of claim 14, furtherincluding an adjuvant.
 16. The kit of claim 14, further includinginstructions for use.
 17. The method of claim 6, wherein saidcomposition further includes a pharmaceutically acceptable carrier. 18.The method of claim 6, wherein said composition further includes anadjuvant.